Method for automatic analysis

ABSTRACT

This invention relates to a method for analyzing the concentration of an analyte in a sample and to automatic analyzing apparatus. The invention will be described herein with particular reference to a method and apparatus for measuring the concentration of glucose or other analytes in blood but is not limited to that use.

This application is a continuation of copending U.S. patent applicationSer. No. 09/970,461 filed on Oct. 2, 2001, now U.S. Pat. No. 6,852,212,which is a continuation of copending U.S. patent application Ser. No.09/502,907, filed Feb. 11, 2000, now U.S. Pat. No. 6,325,917, which is acontinuation of International Application No. PCT/AU98/00642, filed onAug. 13, 1998, which claimed priority from Australian ProvisionalApplication No. PO 8558, filed on Aug. 13, 1997.

FIELD OF THE INVENTION

This invention relates to a method for analyzing the concentration of ananalyte in a sample and to automatic analyzing apparatus. The inventionwill be described herein with particular reference to a method andapparatus for measuring the concentration of glucose or other analytesin blood but is not limited to that use.

BACKGROUND OF THE INVENTION

In our copending applications PCT/AU/00365, PCT/AU/00723, andPCT/AU/00724 (the disclosures of which are incorporated herein byreference) we have described a method for determining the concentrationof an analyte in a carrier. In that method a sample to be analysed isbrought into contact with a reagent containing an enzyme and a redoxmediator in an electrochemical cell. The cell is a thin layer cellcomprising a working electrode spaced apart from a counter electrode bya spacer which ensures that the two electrodes have substantiallyidentical area and predetermined spacing. The spacing between theelectrodes is essentially close so that after a potential is appliedbetween the electrodes, reaction products from the counter electrodemigrate to the working electrode and vice versa, eventually establishinga steady state concentration profile between the electrodes which inturn results in a steady state current.

It has been found that by comparing a measure of the steady statecurrent with the time rate at which the current varies in the currenttransient before the steady state is achieved, the diffusion coefficientof the redox mediator can be measured as well as its concentration. Itcan be shown that over a restricted time range a plot a 1n(i/i.sub.ss−1) vs time (measured in seconds) is linear and has a slope (denoted byS) which is equal to −4p.sup.2 D/L, where “i” is the current at time“t”, “i.sub.ss ” is the steady state current, “D” is the diffusioncoefficient in cm.sup.2 /sec, “L” is the distance between the electrodesin cm and “p” is the constant pi, approximately 3.14159. Theconcentration of reduced mediator present when the potential was appliedbetween the electrodes is given by −2p.sup.2 i.sub.ss /FALS, where “F”is Faraday's constant, A is the working electrode area and the othersymbols are as given above. As this later formula uses S it includes themeasured value of the diffusion coefficient.

Since L and the electrode area are constants for a given cell,measurement of i as a function of time and i.sub.ss enable the value ofthe diffusion coefficient of the redox mediator to be calculated and theconcentration of the analyte to be determined. In our copendingapplication PCT/AU/00724 there are described methods suitable for massproduction of cells having a substantially constant electrode separationL and electrode area A.

Currently glucose in blood samples is measured in pathology laboratoriesand the like by means of apparatus such the YSI blood analyser in whichsuccessive samples are analysed by means of a hollow cylindrical probein which is mounted a silver and a platinum electrode. The face of theprobe is fitted with a three layer membrane. The middle layer containsan immobilised enzyme which is sandwiched between a cellulose acetateand a polycarbonate membrane. The face of the probe, covered by themembrane, is situated in a buffer filled sample chamber into whichsuccessive samples are injected. Some of the sample diffuses through themembrane. When it contacts the immobilised oxidase enzyme it is rapidlyoxidised producing hydrogen peroxide, the glucose forming aglucono-delta-lactone.

The hydrogen peroxide is in turn oxidised at the platinum anodeproducing electrons. A dynamic equilibrium is achieved when the rate ofperoxide production and removal reach a steady state. The electron flowis linearly proportioned to the steady state peroxide concentration andtherefore to the concentration of the glucose.

The platinum electrode is held at an anodic potential and is capable ofoxidising many substances other than hydrogen peroxide. To prevent thesereducing agents from contribution to sensor current, the membranecontains an inner layer consisting of a very thin film of celluloseacetate. This film readily passes hydrogen peroxide but excludeschemical compounds with molecular weights above approximately 200. Theacetate film also protects the platinum surface from proteins,detergents, and other substances that could foul it. However thecellulose acetate film can be penetrated by compounds such as hydrogensulphide, low molecular weight mercaptans, hydroxylamines, hydrozines,phenols and analytes.

In use, the sample (or a calibration standard) is dispensed in to thechamber, diluted into 600 microliters of buffer, and then a measurementis made by the probe. The sensor response increases and then reaches aplateau when a steady state is reached. After several seconds a bufferpump flushes the chamber and the sensor response decreases.

The apparatus monitors the base line current. If it is unstable a bufferpump will continue to flush the sample chamber with buffer. When astable base line is established an automatic calibration is initiated.The apparatus calibrates itself for example after every five samples or15 minutes. If a difference of more than 2% occurs between the presentand previous calibration. the apparatus repeats the calibration.Recalibration also occurs if the sample chamber temperature drifts bymore than 1.degree. C.

The apparatus described suffers from a number of disadvantages. Firstly,a high proportion of its time in use is spent in performing calibrationsrather than analysis. Furthermore the consumption of buffer andcalibrating solutions is a substantial cost. Another disadvantage isthat as the enzyme membrane ages, a graph of reading versusconcentration becomes non-linear. It would be highly desirable toprovide apparatus which is able to make measurements of the kinddescribed with improved speed, efficiency, and at lower running cost.

SUMMARY OF THE INVENTION

The present invention generally provides improved methods and apparatusfor automatically analyzing samples which avoids or ameliorates at leastsome of the disadvantages of prior art. In one aspect, a method isprovided that accounts for slow processes occurring in a test.

In one embodiment, a method for estimating the concentration of areduced (or oxidised) form of a redox species in a liquid is disclosed.The method can includes the steps of:

-   -   (1) contacting an area of a first electrode with a sample of        predetermined volume of the liquid,    -   (2) contacting the sample with an area of a second electrode        spaced apart from the first,    -   (3) applying a potential between the electrodes while the        electrodes are sufficiently closely spaced that reaction        products formed at each electrode diffuse to the other electrode        while the potential is applied,    -   (4) reversing the potential between the electrodes,    -   (5) measuring or estimating a value indicative of the change in        current as a function of time and a value indicative of the        steady state current, and    -   (5) determining from said volume, said current as a function of        time, and said steady state current, the concentration of        reduced (or oxidised) form of the species in the liquid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention will now be described by way ofexample only with reference to the accompanying drawings wherein:

FIG. 1 is a schematic diagram showing in cross-section a firstembodiment of apparatus according to the invention;

FIG. 2 is a schematic diagram showing, in enlarged cross-section, asample droplet between two electrodes;

FIG. 3 is a schematic diagram showing in cross-section a secondembodiment of apparatus according to the invention;

FIG. 4 is a schematic diagram of a third embodiment of apparatusaccording to the invention in side elevation; and

FIG. 5 shows the embodiment of FIG. 4 in end elevation, viewed on line5-5 of FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

By way of example a first embodiment of apparatus according to theinvention will be described.

With reference to FIG. 1 there is shown schematically an automaticanalyser for measuring glucose in blood samples. The apparatus comprisesa flexible first electrode 1 consisting of a palladium layer 2 formed ordeposited onto a flexible carrier 3 (for example a 100 micron PET film)preferably by sputter coating to a thickness of for example 100-1000angstrom. Electrode 1 is fed into the analyser from a roll (notillustrated) in the form of a tape.

First electrode 1 is provided on palladium surface 2 with an enzyme anda redox mediator. These may be selected (without limitation) from thesystems in table 1 and in the present example a GOD enzyme andferricyanide mediator are used. The enzyme and redox mediator may beprinted in predetermined quantities at predetermined intervals on thefirst electrode surface as a dried reagent coating 4.

Electrode 1 is driven by means not shown in the drawings through asample station “S” at which a precise volume of a sample 1 is placed asa droplet 5 on a reagent coating 4 on electrode surface 1, for example,by means of an automatic pipette 6. Less preferably, predeterminedquantities of enzyme and redox mediator may be combined with the samplebefore or after deposition of the droplet on the electrode.

A second electrode 11 which in the present example is of similarconstruction to the first electrode, and comprising a palladium layer 12sputter coated onto a flexible PET carrier 13, is then brought intoclosely spaced relationship with electrode 1 and into contact with adroplet 5. The droplet wets both palladium surfaces 1 and 10 and adoptsa substantially cylindrical configuration between the two electrodes asmore clearly illustrated in FIG. 2. The droplet is bounded intermediateelectrodes 1, 2 by a liquid/gas interface 14.

An electric potential is then applied to the two electrodes (by meansnot illustrated in FIG. 1) via contacts.

As described in our co-pending applications PCT/AU96/00723 andPCT/AU96/00724, the potential between the electrodes is set such thatthe rate of electro-oxidation of the reduced form of the species (or ofelectro-reduction of the oxidised form) is diffuision controlled.Because the working and counter electrodes are placed in very closeproximity (about 0.5 mm apart or less) ferricyanide that is generated atthe counter electrode has time to reach the working electrode andcontribute to the current at the working electrode. That is, aferricyanide molecule can be reduced at the counter electrode toferrocyanide, and can then diffuse to the working electrode, where itwill be re-oxidised to ferricyanide. This situation results in adecreasing current at short times that steadies to reach a constantvalue at longer times (the steady state current). This steadying of thecurrent occurs because a constant stream of ferrocyanide is beingsupplied to the working electrode from the counter electrode. Thismechanism is quite distinct from that which occurs in a Cottrell devicein which the electrodes are separated so that ferricyanide that resultsfrom the reduction of ferricyanide at the counter electrode does notinfluence the observed current.

In the present cell the steady state current is given by $\begin{matrix}{l_{\#} = \frac{2{DFAC}_{0}}{L}} & (1)\end{matrix}$

wherein i.sub.ss is the steady state current, D is the diffusioncoefficient, F is the Faraday constant, A is the area of the electrode,C₀ is the concentration of the analyte (ferricyanide) and L is theseparation of the electrodes.

The current i at time t is given by the equation: $\begin{matrix}{i = {i_{xx}\left( {1 + {2{\sum\limits_{n = 1}^{\infty}\quad{\mathbb{e}}^{\frac{{- 4}p^{2}n^{2}{Dt}}{L^{2}}}}}} \right)}} & (2)\end{matrix}$where p is pi.

At longer times the higher exponential terms in equation 2 can beignored. Therefore equation 2 can be approximated by equation 3 fortimes greater than a certain value $\begin{matrix}{i = {i_{ss}\left( {1 + {2{\exp\left( \frac{{- 4}p^{2}{Dt}}{L^{2}} \right)}}} \right)}} & (3)\end{matrix}$

If it is assumed that equation 2 can be approximated by equation 3 whenthe second exponential term in equation 2 is 1% of the first exponentialterm, equation 3 is valid for times greater than$i = {\frac{0.0389L^{2}}{D}.}$

It will be understood that Equation 3 can be transformed to give:$\begin{matrix}{{\ln\left( {\frac{l}{iss} - 1} \right)} = {{\ln(2)} - {4p^{2}\frac{Dt}{L^{2}}}}} & \left( 4^{\prime} \right)\end{matrix}$

So a plot of the left hand side of equation (4′) versus time will give astraight line with new $\begin{matrix}{{slope} = {{- 4}p^{2}\frac{Dt}{L^{2}}}} & \left( 5^{\prime} \right)\end{matrix}$

Combining equations (1) and (5′) gives $\begin{matrix}{{Co} = {- \frac{2p^{2}{iss}}{FVslope}}} & \left( 6^{\prime} \right)\end{matrix}$where V=AL is the volume of the drop of sample pipetted onto the tape.Since the parameters “slope” and “iss” are measured in the test and pand F are universal constants, to measure the concentration of theanalyte derived from the test (Co) it is only required to know thevolume of the sample pipetted. Since this can be done very accurately itis possible to have a very accurate measure of Co without the need forany other calibration of the system. Significantly, neither the spacingbetween the electrodes nor the electrode area wetted need be known.

The exact shape adopted by the droplet in contact with the twoelectrodes is not important.

If desired the chemistry of successive electrode locations could bedifferent one from another so that a multiplicity of different testscould be performed on successive pipetted volumes of sample placed atsuccessive electrode locations. In a second embodiment as shown in FIG.3 which corresponds to the portion of FIG. 1 upstream from samplestation S, the first electrode is provided with an overlying layer 7 forexample of a thin PET film from which apertures have been punched todefine wells 8 into which chemical reagents 4 can be placed. and whichserved to define the locations at which reagents have been placed and/orto protect the reagents prior to use. In this case electrode 1 isconveniently supplied to the apparatus from a roll having predeterminedquantities of chemical reagents in the wells in dried form and protectedfrom contamination prior to use by being sandwiched between layers ofthe roll. The chemical reagents are only used once and therefore can bemore easily protected against deterioration than is possible with priorart. In the above described embodiment the sample droplet 5 is not“contained” by a cell although it may be deposited and located within awell 8. When a well-defining layer 7 is employed it may be adhered tothe electrode surface or electrode carrier or it may merely be anon-adhered spacer layer.

It is not necessary for the upper electrode layer 11 to come intocontact with the top surface of the well-defining layer 7. The volume ofsample pipetted is such that the height of drop 5 is equal to orpreferably greater than the thickness of the well-defining layer 7. If alayer 7 is used to define a well 8, it is undesirable for the samplevolume to run to the sides of the well. It is sufficient that the sampleis a known volume and wets both electrodes preferably forming asubstantially cylindrical shape therebetween.

It will also be understood that the well-defining layer 7 can bereplaced with a porous layer for example a porous paper, non-woven mesh,or felt, or a porous membrane, which acts to immobilise the samplespatially with respect to the electrode layers and to hold the reagentsin place and in this case the second electrode will contact the surfaceof the porous layer immobilising the volume.

It will be understood that use of a porous or well-defining layer 7 isoptional and that in other embodiments of the invention a layer 7 isunnecessary it being sufficient for a drop of sample to be pipetted ontoa metal layer 2 and for an upper metal layer 12 to be brought intocontact with a sample drop of predetermined volume, upper metal layer 12being desirably but not essentially at a predetermined spacing from thelower metal layer 1.

It will also be understood that the metal layer tapes or bands need notbe travelling in the same direction. For example, one metallisedelectrode layer may be proceeding transversely of the other, each tapebeing advanced after each measurement to expose a fresh lower and freshupper electrode surface and fresh reagent at the sample filling station.In each case the resulting current is measured as a function of timewhile the electrodes are in contact with a sample drop of predeterminedvolume.

Continuous band electrodes are preferred. These may either be disposedof after use or may be passed through a washing station and then reused,if desired after reprinting with reagents.

In preferred embodiments of the invention predetermined quantities ofreagent are placed on one or both of the electrodes by metering devicesfor example an ink jet print-head upstream of sample station 3 and may,but need not, be dried prior to contact with the sample. A reagentapplication system may be a part of the apparatus, or the apparatus maybe adapted to receive electrodes in roll or other form pretreated withthe desired reagents at another location or plant.

It will be understood that one or both electrodes need not be acontinuous band but may, for example, be in the form of a retractableprobe. The second electrode could be a disposable probe lowered intocontact with a droplet on a first electrode and then withdrawn aftercurrent measurements are completed. Likewise the first electrode neednot be in the form of a tape. The first electrode could for example bemounted to a carousel or be in the form of a rotating disc. Although itis preferred to use disposable electrode surfaces, the method may beapplied with reusable electrodes washed in between successive uses. Byway of example, there is shown schematically in FIG. 4 an automaticanalyzing apparatus comprising a first electrode 1 in the form of afirst disc driven intermittently in rotation about a first horizontalaxis. A second electrode 11 is driven intermittently and synchronouslywith the first disc in rotation about a horizontal axis parallel to thefirst axis. Electrodes 1 and 11 are spaced apart at their edge at theclosest point of approach. Sample drops 5 of precisely predeterminedvolume are deposited on the first electrode at intervals by a pipettingdevice 6 in synchronization with the disc rotation. Reagents 4 areprinted on the second electrode at corresponding intervals by means of aprinting roll 16 and are dried in situ, for example by an air blower(not shown in the drawing).

In use, as electrode 1 rotates, a drop 5 travels to a position where itcomes into contact with the second electrode and with the reagentsprinted thereupon. While both discs are stationary with the droplet incontact with each electrode, a potential is applied between theelectrodes and the current measured as previously discussed. During thistime the reagent(s) dissolve in the sample and after the necessarymeasurements have been made, both electrodes are indexed to a new angleof rotation. The surfaces used for the analysis are washed clean bysprays 14, into drained sumps 15 and ready for reuse.

Apparatus according to the invention requires very much smaller samplesthan are required with the YSI device and because the chemical reagentscan be better protected until used and more accurately metered, theapparatus provides greater accuracy and speed at reduced cost.

In another embodiment of the invention the current can be followed withtime after a potential has been applied between the electrodes until apredetermined time or state has been reached. The sign of the appliedpotential would then be reversed and analysis performed similar to thatgiven above except with equations (3) and (4) being replaced with$\begin{matrix}{i = {{iss}\left( {1 + {4{\exp\left( {{- 4}p^{2}\frac{Dt}{L^{2}}} \right)}}} \right)}} & (7) \\{{\ln\left( \frac{i}{{iss} - 1} \right)} = {{\ln(4)} - {4p^{2}\frac{Dt}{L^{2}}}}} & (8)\end{matrix}$

This protocol has the advantage of being able to allow for slowprocesses occurring in the test. This can be done by:

-   -   a) waiting for the current to change by less than a        predetermined amount per second before reversing the potential,        such that any slow processes which effect the measurement are        substantially complete, or    -   b) using the change in the current with time before the        potential is reversed to compensate for the slow processes        occurring (as has been described in our earlier patent        applications in relation to cells having a predefined electrode        separation and area).

Although the invention has been described with reference to palladiumelectrodes, the electrodes can be of other suitable metals such asdescribed for example in our earlier applications referred to herein.One electrode may be different from the other. The electrodes may besupported by PET as exemplified above or by other suitable insulatingmaterials or may be self-supporting. If supported on an insulating film,it is preferred, but not essential, that the metals be deposited on thefilm by sputter coating. Electrical contact for the application of apotential and/or for the measurement of current may be by any suitablemeans including clamping engagement with one end of the electrode if inthe form of a tape, or by means of suitable rolling contacts, orspringloaded contacts, or the like. The application of the electricalpotential; the measurement of current; the calculation of theconcentration of analyte; the synchronous control of the movement of oneelectrode with respect to the other and with the deposition of sampledroplets and, if required, with the deposition of reagents may becontrolled by a microprocessor or the like and the results may beprinted, displayed, and/or otherwise recorded by means which arewell-known to those skilled in the control arts.

As will be appreciated by those skilled in the art from the teachinghereof the features of one embodiment may be combined with those ofanother and the invention may be embodied in other forms withoutdeparting from the concepts herein disclosed.

Parent applications application Ser. No. 09/970,461 filed on Oct. 2,2001, now U.S. Pat. No. 6,852,212, and application Ser. No. 09/502,907,filed Feb. 11, 2000, now U.S. Pat. No. 6,325,917 are hereby incorporatedby reference in their entirety. TABLE 1 REDOX MEDIATOR ANALYTE ENZYMES(OXIDISED FORM) ADDITIONAL MEDIATOR Glucose GDpqq Ferricyanide2,6-dimethyl-1,4-benzoquinone 2,5- Glucose (NAD Glucose dehydrogenaseand diaphorase Ferricyanide dichloro-1,4-benzoquinone or phenazinedependent) Cholesterol esterase and cholestrol Ferricyanide ethosulfateCholesterol oxidase HDI cholesterol Cholestrol esterase and cholesterolFerricyanide 2,6-dimethyl-1,4-benzoquinone 2,5- oxidasedichloro-1,4-benzoquinone or phenazine ethosulfate TriglyceridesLipoprotein lipase, glycerol kinase, and Ferricyanide or phenazineethosulphate Phenazine methosulfate glycerol-3-phosphate oxidase LactateLactate oxidase Ferricyanide 2,6-dichloro-1,4-benzoquinone LactateLactate dehydrogenase and diaphorase Ferricyanide, phenazineethosulfate, or phenazine methosulfate Lactate Diaphorase Ferricyanide,phenazine ethosulfate, or dehydrogenase phenazine methosulfate PyruvatePyruvate oxidase Ferricyanide Alcohol Alcohol oxidase PhenylenediamineBilirubin Bilirubin oxidase 1-methoxy-phenazine methosulfate Uric acidUricase Ferricyanide

1. A method for estimating the concentration of a reduced or oxidizedform of a redox species in a liquid comprising the steps of: (1)contacting an area of a first electrode with a sample of predeterminedvolume of the liquid, (2) bringing a second electrode into a closelyspaced relationship with the first electrode, thereby contacting thesample with an area of the second electrode spaced apart from the firstelectrode, wherein step (2) is conducted after step (1), (3) applying apotential between the electrodes while the electrodes are sufficientlyclosely spaced such that reaction products formed at each electrodediffuse to the other electrode while the potential is applied, (4)reversing the potential between the electrodes, (5) measuring orestimating a value indicative of the change in current as a function oftime and a value indicative of the steady state current, and (6)determining from said value, said current as a function of time, andsaid steady state current the concentration of reduced or oxidized formof the species in the liquid sample.
 2. The method according to claim 1,wherein the step of reversing the potential occurs after waiting for thecurrent to change by less than a predetermined amount per time.
 3. Themethod according to claim 1, further comprising the step of the changein current as a function of time before the potential is reversed tocompensate for a slow chemical reaction step.
 4. The method according toclaim 1, wherein at least one of the electrodes includes a reagentpositioned thereon.
 5. The method according to claim 4, wherein thesample contacts the reagent during step (1).
 6. The method according toclaim 4, wherein the sample contacts the reagent during step (2).
 7. Themethod according to claim 4, wherein the sample is deposited on thereagent.
 8. The method according to claim 1, wherein at least oneelectrode is covered with a layer which serves to define wells on theelectrode surface.
 9. The method according to claim 1, wherein at leastone of the electrodes is in the form of a continuous strip.
 10. Themethod according to claim 1, wherein a pipette deposits the sample onthe first electrode.
 11. The method according to claim 1, wherein thefirst electrode further comprises a porous medium wherein thepredetermined volume is immobilized.
 12. The method according to claim1, further comprising the step of depositing one or more reagents on oneof the electrodes prior to placing the sample on the electrode.
 13. Themethod according to claim 1, wherein at least one of the first andsecond electrodes includes at least one well.
 14. The method accordingto claim 1, wherein at least one of the first electrode and the secondelectrode is in the form of a disposable probe.
 15. The method accordingto claim 1, wherein at least one of the first electrode and the secondelectrode is in the form of a retractable probe.
 16. The methodaccording to claim 1, wherein the first electrode comprises a firstmetallized electrode layer, wherein the second electrode comprises asecond metallized electrode layer, and wherein the first metallizedelectrode layer travels transversely of the second electrode layer. 17.The method according to claim 1, wherein the first electrode comprises afirst metallized electrode layer, wherein the second electrode comprisesa second metallized electrode layer, and wherein the first metallizedelectrode layer travels in a same direction as the second electrodelayer.
 18. The method according to claim 1, wherein the sample ofpredetermined volume is a droplet deposited on one of said electrodes.19. The method according to claim 18, wherein the droplet is heldbetween the two electrodes by surface tension.